There are two main types of restriction endonucleases ("restriction enzymes"), type I and type II. Both types cleave double-stranded DNA molecules. Type I restriction enzymes recognize a specific DNA sequence, but cleave the DNA molecule at varied sites removed from that sequence. In contrast, type II restriction enzymes recognize a specific DNA sequence and cleave DNA molecules at or adjacent specific sequences (a "restriction site"). Type II restriction enzymes are therefore particularly useful in biological techniques such as molecular cloning, genetic mapping and DNA sequence analysis. See generally Molecular Cloning: A Laboratory Manual, J. Sambrook et al. (2nd Ed. 1989).
However, type II restriction enzymes may be quite unstable, especially in the presence of Mg.sup.+2, which is a cofactor for some proteases. If activated, proteases can degrade a restriction enzyme. Mg.sup.+2 is also a nutrient source for some bacteria that can contaminate and inactivate an enzyme preparation. Also, conformational and other changes to restriction enzymes can occur during improper storage. Thus, methods have been developed to reduce storage problems associated with restriction enzymes.
One well known prior art method of storage is freeze-drying. In this method, an aqueous solution of the enzyme in a conventional storage buffer and in the presence of a cryoprotectant is first frozen, typically to -40.degree. to -50.degree.. Water is then removed from the biological solution and the residual material becomes more concentrated until the material crystallizes. Ice is then removed by sublimation under vacuum. When the last traces of residual moisture are removed, a dry crystalline powder remains. An active enzyme may be reconstituted from this powder.
Unfortunately, exposure of a freeze-dried product to ambient temperatures can result in significant enzyme activity loss. Additionally, restriction enzymes are often not completely freeze-stable. Further, the freeze-drying cycle may take several days and the activity of the reconstituted enzyme may not be very reproducible.
The "freeze/thaw" storage method involves mixing the enzyme with a cryoprotectant, freezing and storing, usually below -50.degree. C. and sometimes in liquid nitrogen. The enzyme is then thawed immediately before use. Some enzymes will not survive a freezing and thawing cycle. Further, this technique can be expensive (especially with respect to transportation to customers).
Another technique (the one currently in commercial widespread use) involves storing an enzyme solution at -20.degree. C. This type of refrigerated storage for restriction enzymes usually involves the addition of the cryoprotection additive glycerol to depress the freezing point and avoid freezing the enzyme. Restriction enzymes are usually stored in 50% glycerol.
A significant problem with this type of storage of restriction enzymes is that the presence of glycerol in a restriction enzyme reaction can lead to what is known as specificity relaxation (such as "star activity"). In this regard, when restriction enzyme digestions are done in the presence of glycerol, the specificity of the enzyme for a particular restriction site becomes relaxed and DNA is no longer cleaved only at that restriction site. The lack of enzyme specificity may lead to confusion as to which DNA fragments are the result of true restriction enzyme cleavage. It is generally understood that a glycerol concentration of greater than 5% can create a significant possibility of star activity in an enzymatic digest. High enzyme concentration in the DNA cleavage reaction is also known to contribute to star activity, even in the absence of glycerol. To date, the art has often sought to minimize specificity relaxation by using lower than desired concentrations of enzyme and/or longer than desired incubation times.
Another method of storing restriction enzymes involves forming a "glass" with the restriction enzyme embedded within it. A stabilizer (usually a carbohydrate) is used that is capable of forming a glass and does not interfere with DNA cleavage reactions. In the glass, the restriction enzyme is virtually immobilized and stable, even at room temperature. However, when this storage technique was used, water and appropriate salts and reagents had to be added to the glassified enzyme to create a solution capable of cleaving DNA.
The prior glassification method is therefore limited in that it required the lab worker to add the various reagents (especially Mg.sup.+2) to activate the restriction enzyme after storage. Also, high enzyme concentrations in the cleavage reaction still would lead to star activity, even in the absence of glycerol.
A need therefore existed for an improved system of storing restriction enzymes so that the enzymes would remain stable during long-term storage at room temperature, could be activated upon reconstitution with only water and substrate DNA (without the need to separately add other chemicals), and so that star activity could be reduced even for high enzyme concentrations.